A majority of protein kinases share a common DFG (Asp-Phe-Gly) motif in the ATP site which transitions between two distinct conformations in response to phosphorylation of the contiguous activation loop: the active DFG-in and the inactive DFG-out states. Kinase inhibitors that bind only to the DFG-in state often suffer from a lack of target specificity, as the ATP site is wide open and able to accommodate diverse chemical scaffolds. By contrast, inhibitors able to induce and stabilize the DFG-out conformation are considered superior, as they render the active site architecture incompatible with substrate binding, resulting in enhanced potency and target selectivity. The clinical success of imatinib (Gleevec) as an inhibitor of Abl kinase (Nagar et al., (2002) Cancer Res 62:4236-4243) is attributed in large part to this distinct mode of action (Seeliger et al., (2009) Cancer Res 69:2384-2392) and has spurred the design of DFG-out inhibitors for other kinases, including MAP (Angell et al., (2008) Bioorg Med Chem Lett 18:4433-4437), JNK2 (Kuglstatter et al., (2010) Bioorg Med Chem Lett 20:5217-5220), Nek2 (Solanki et al., (2011) J Med Chem 54:1626-1639), and Eph receptor tyrosine kinase (Choi et al., (2009) Bioorg Med Chem Lett 19:4467-4470). However, all known Aurora kinase inhibitors, such as the aforementioned chemical probe VX680, are DFG-in inhibitors. Although the DFG motif is highly conserved among protein kinases, the mechanism by which small molecules induce the DFG flip is not well understood. Small molecules able to induce large conformational changes in the target enzyme have potential as superior lead compounds in drug discovery, as the altered structure of the dead-end complex is less suited for efficient interaction with substrate. This concept has led to the design of some of the most clinically successful kinases inhibitors to date. Imatinib and sorafenib stabilize the DFG-out conformation by establishing a bridging network of hydrogen bonds between the amide/urea inhibitor core and both a conserved glutamate side chain within the C-helix and the main chain amide of the DFG aspartate residue (Dietrich et al., (2010) Bioorg Med Chem 18:5738-5748). Molecular dynamics simulations were used to elucidate and propose a mechanism for the DFG-out conformation in MAPK p38a, in which the phenylalanine of the DFG motif is forced by the inhibitor from its hydrophobic pocket in the DFG-in (active state) to the solvent-exposed DFG-out (inactive state), triggering an overall rearrangement of the activation loop (Filomia et al., (2010) Bioorg Med Chem 18:6805-6812). However, the knowledge gained from these structures did not translate into an applicable method for the rational design of DFG-out inhibitors of other kinases.
DFG-out inhibitors of Aurora A utilizing a bisanilinopyrimidine scaffold are disclosed herein. A series of co-crystal structures established that electronegative and electron-withdrawing substituents, directed at the N-terminally flanking residue Ala273, yielded highly potent DFG-out inhibitors able to induce and stabilize a unique “DFG-out/loop-in” conformation. The data suggest an unprecedented mechanism of action, by which induced-dipole forces disrupt the charge distribution along the DFG peptide, causing the DFG to unwind. As the ADFG sequence is highly conserved among kinases, the strategy employed here to inhibit Aurora A may be applicable to other kinases as well.